Genome-wide and protein kinase-focused RNAi screens reveal conserved and novel damage response pathways in Trypanosoma brucei

All cells are subject to structural damage that must be addressed for continued growth. A wide range of damage affects the genome, meaning multiple pathways have evolved to repair or bypass the resulting DNA lesions. Though many repair pathways are conserved, their presence or function can reflect the life style of individual organisms. To identify genome maintenance pathways in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens to identify genes important for survival following exposure to the alkylating agent methyl methanesulphonate. Amongst a cohort of broadly conserved and, therefore, early evolved repair pathways, we reveal multiple activities not so far examined functionally in T. brucei, including DNA polymerases, DNA helicases and chromatin factors. In addition, the screens reveal Trypanosoma- or kinetoplastid-specific repair-associated activities. We also provide focused analyses of repair-associated protein kinases and show that loss of at least nine, and potentially as many as 30 protein kinases, including a nuclear aurora kinase, sensitises T. brucei to alkylation damage. Our results demonstrate the potential for synthetic lethal genome-wide screening of gene function in T. brucei and provide an evolutionary perspective on the repair pathways that underpin effective responses to damage, with particular relevance for related kinetoplastid pathogens. By revealing that a large number of diverse T. brucei protein kinases act in the response to damage, we expand the range of eukaryotic signalling factors implicated in genome maintenance activities

Distinct gene expression patterns in vector-residing Leishmania infantum identify parasite stage-enriched markers.

BACKGROUND:Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking. METHODOLOGY/PRINCIPAL FINDINGS:We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum. CONCLUSIONS:Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies

Molecular modeling and structure–activity relationships studies of bioisoster hybrids of N-acylhydrazone and furoxan groups on cruzain

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Chagas disease is a very important neglected disease and millions of people live in endemic areas at the risk of acquiring the infection. Due to scarce and ineffective current chemotherapy, the introduction of new drugs on therapeutics is highly necessary. Bioisoster hybrids derivatives were previously designed, synthesized, and assayed in terms of trypanocidal activity and permeability. Structure activity-relationships were performed with a set of N-acylhydrazone and furoxan derivatives aiming at identifying if the enzyme cruzain might be the target of the system. In addition, lowest unoccupied molecular orbital analysis and docking studies of the two most promising compounds were carried out with the purpose of elucidating the key properties and interactions between the ligands and cruzain. The analysis of cruzain inhibition tests showed that the position of the nitro group is important for the compounds inhibitory activity. The results lead to the conclusion that cruzain may not be the only target. This hypothesis was advanced because the most active in Trypanosoma cruzi, compound 6, is not the most effective in cruzain tests. Notwithstanding, this compound was considered the most promising hit for further in vivo studies, as it did not show toxicity in cycle cell tests.Chagas disease is a very important neglected disease and millions of people live in endemic areas at the risk of acquiring the infection. Due to scarce and ineffective current chemotherapy, the introduction of new drugs on therapeutics is highly necessary26760769COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)sem informaçãosem informaçãosem informaçãoThe authors thank Kerly Fernanda Mesquita Pasqualoto, PhD, from Instituto Butantan, Sao Paulo, for allowing the use of MOLSIM 3.2 software, which is under her responsibility, as well as Prof. Sandro Rogério de Almeida and Renata Chaves Albuquerque, from

A composite recombinant salivary proteins biomarker for Phlebotomus argentipes provides a surveillance tool post-elimination of visceral leishmaniasis in India.

peer reviewedIncidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the post-elimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Ph. papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The Receiver Operating Characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >0.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts post-VL elimination

A Composite Recombinant Salivary Proteins Biomarker for Phlebotomus argentipes Provides a Surveillance Tool Postelimination of Visceral Leishmaniasis in India

Incidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the postelimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Phlebotomus papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after VL elimination

A composite recombinant salivary proteins biomarker for Phlebotomus argentipes provides a surveillance tool postelimination of visceral leishmaniasis in India

peer reviewedIncidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the post-elimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Ph. papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The Receiver Operating Characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >0.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts post-VL elimination

Loss of AUK2 results in nuclear DNA damage.

<p><b>A.</b> Western blot analysis of γH2A in two <i>auk2</i> -/- mutants clones (CL1 and CL2) relative to an <i>AUK</i>+/- heterozygous mutant and wild type cells (WT427). Whole cell lysates were probed with anti-γH2A (below) and anti-EF1α (above; loading control) antisera. The graph shows levels of γH2A after normalisation by EF1α: γH2A levels in WT cells were set at 1 and fold change in the mutants relative to WT is shown. Data points represent means and SEM (n = 3). <b>B.</b> Immunofluorescence (IF) of RAD51 foci formation. Cells were harvested, fixed and RAD51 localised with anti-RAD51 antiserum. Representative IF images of <i>auk2</i>-/- mutants are shown in which DAPI stained DNA is in blue and RAD51 in red (cell morphology is shown by differential contrast imaging); the scale bar = 10 μm. The graph shows the percentage of WT cells with detectable RAD51 foci compared with <i>AUK2</i>+/- mutants and two <i>auk2</i>-/- clones. Cells with RAD51 foci are represented as a percentage of the total population of cells counted (n >200). Data points represent the mean from three independent experiments; errors bars show SEM. * denotes a significant difference from WT (P<0.05, Mann Whitney U test).</p

Analysis of the MMS RIT-seq screen.

<p><b>A, B.</b> Scatter plots showing the ratio of mapped RNAi target-specific reads for every gene (grey dots) in the RNAi-induced, MMS-treated population relative to the RNAi-induced, untreated population (MMS+/MMS-); gene location within the 11 megabase chromosomes is shown and dotted lines indicate 2-fold increase and decrease in MMS+/MMS- ratio. Genes are highlighted with roles in (A) homologous recombination (HR, red), mismatch repair (MMR, blue) and nucleotide excision repair (NER, green), or in (B) intraflagellar transport (IFT, red), mitochondrial replication (Mito rep, blue) and encoding histones (green). <b>C.</b> A pie chart of the distribution of all genes displaying an MMS+/MMS- ratio of less than 0.5, excluding 44 genes predicted to be VSGs. Hypothetical and hypothetical unlikely denotes genes for which there are currently no homology-predicted functions. Unknown denotes genes with homology-predicted functions that cannot be readily associated with the response to MMS damage. Finally, genes in seven classes of predicted functions with putative roles in responding to MMS are detailed. <b>D.</b> GO terms, within two headings, which show significantly increased frequency in the MMS+/MMS- <0.5 gene set relative to the whole GO gene set (IDs and further analysis are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s003" target="_blank">S3 Table</a>).</p

Damage response protein kinases predicted by whole-genome MMS RIT-seq.

<p><b>A.</b> Scatter plot of MMS+/MMS- read depth ratios for all <i>T</i>. <i>brucei</i> genes (grey dots). Protein kinase (PK) genes are highlighted in red and individual genes are further identified (arrows) by gene IDs and names, if known (further details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s002" target="_blank">S2 Table</a>). <b>B.</b> Nine PK genes, including PK family and name, present in the MMS+/MMS- <0.5 gene set are listed. <b>(C</b> displays read mapping profiles for selected PKs (red) after RNAi and growth with (MMS+) or without (MMS-) 0.0003% MMS (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.g005" target="_blank">Fig 5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s011" target="_blank">S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s012" target="_blank">S6</a> Figs for more detail).</p